Abstract Shiga toxin-producing E. coli are a major cause of foodborne illness in the United States and infect over 100,000 individuals annually. These bacteria produce two types of Shiga toxin (STx): STx1 and STx2. The toxins are formed by the association of an A subunit, which kills host cells by blocking protein synthesis, with a pentameric B-subunit, which mediates trafficking from the cell surface to the cytosol. There are no treatments for disease caused by Shiga toxin-producing E. coli, including for life-threatening renal complications. As STx1 and STx2 must traffic to the cytosol to induce toxicity, there is considerable interest in designing inhibitors of toxin trafficking for therapy. In 2012, we identified the metal manganese to be a potent inhibitor of STx1 trafficking that protected mice against STx1-induced lethality. However, disease severity correlates with STx2 production. Yet, mechanisms of STx2 trafficking have not been elucidated and approved inhibitors of STx2 trafficking are not available. Therefore, our recent efforts focused on STx2 transport. We discovered that, in host cells, both STx1 and STx2 used a conserved domain (?4-?5 loop of their B- subunits) to avoid lysosomal degradation and directly traffic to the Golgi from early endosomes. The ?4-?5 loop of STx1 B-subunit interacted with a host protein, GPP130, which functioned as the endosomal receptor for STx1 and chaperoned the toxin to the Golgi from early endosomes. Manganese blocked STx1 trafficking by inducing GPP130 degradation. In contrast, STx2 trafficking was GPP130 independent (and Mn insensitive) because residues required for GPP130 binding were not conserved in its B-subunit. Despite the molecular difference, the fact that STx1 and STx2 use a common structural motif to undergo early endosome-to-Golgi transport raises the possibility that the underlying mechanisms may have conceptual similarities. We hypothesize that, similar to the GPP130-STx1 paradigm, the ?4-?5 loop of STx2 B-subunit interacts with a host protein, which acts as the endosomal receptor for STx2. This interaction allows STx2 to traffic to the Golgi from early endosomes, and thereby avoid lysosomal degradation. In this exploratory proposal, we will test this hypothesis by identifying host factors that are required for the early endosome-to-Golgi transport of STx2 and subsequently determining whether any of these factors function as the endosomal STx2 receptor. To achieve our experimental goals, we will combine a high-throughput proteomic screen in Aim 1 with a genome- wide siRNA screen in Aim 2. As Mn-induced degradation of GPP130, the STx1 endosomal receptor, is remarkably effective in protecting against STx1 toxicity, identification of the endosomal receptor for STx2 is expected to provide an ideal molecular target for design of STx2 transport inhibitors. Thus, this exploratory proposal has the potential to make far-reaching contributions towards the development of a treatment for disease caused by Shiga toxin-producing E. coli, based on blocking STx2 trafficking.